Protein kinase inhibitors

ABSTRACT

The Pyrazolo[1,5-a]pyrimidine derivatives represented by formula I and their pharmaceutically acceptable salts exhibit excellent kinase inhibiting activity. Drugs comprising the compounds as effective ingredients are therefore expected to be useful as therapeutic or prophylactic agents for a protein kinase mediated disorder in which kinase is implicated, such as inflammatory disease, autoimmune disease, destructive bone disorder, cancer and/or tumour growth.

FIELD OF THE INVENTION

The present invention relates to the use of certain compounds in theinhibition of protein kinases, in particular inhibitors ofmitogen-activated protein kinase (MAPK) family, more particularlyserine/threonine kinases, mitogen-activated protein kinase-activatedprotein kinase 2 (MAPKAP-K2). Their use in medicine and particularly inthe prevention and/or treatment of inflammatory and neurologicaldisorders is described.

BACKGROUND ART

Protein kinases are a family of enzymes that catalyse thephosphorylation of hydroxyl groups in proteins. Approximately 2% of thegenes encoded by the human genome are predicted to encode proteinkinases. The reversible phosphorylation of specific tyrosine, serine, orthreonine residues on a target protein can dramatically alter itsfunction in several ways including activating or inhibiting enzymaticactivity; creating or blocking binding sites for other proteins;altering subcellular localisation or controlling protein stability.Consequently protein kinases are pivotal in the regulation of a widevariety of cellular processes, including metabolism, cell proliferation,differentiation and survival. Of the many different cellular functionsknown to require the actions of protein kinases, some represent targetsfor therapeutic intervention for certain disease states.

It is known that several diseases can arise from, or involve, aberrantprotein kinase activity. In humans, protein tyrosine kinases are knownto have a significant role in the development of many disease statesincluding diabetes, cancer and have also been linked to a wide varietyof congenital syndromes. Serine threonine kinases also represent a classof enzymes, inhibitors of which are likely to have relevance to thetreatment of cancer, diabetes and a variety of inflammatory disorders.Modulation of protein kinase activity therefore represents an attractivearea for the design of new therapeutic agents.

Three potential mechanisms for inhibition of protein kinases have beenidentified thus far. These include a pseudo-substrate mechanism, anadenine mimetic mechanism and the locking of the enzyme into an inactiveconformation by using surfaces other than the active site. The majorityof inhibitors identified/designed to date act at the ATP-binding site.Such ATP-competitive inhibitors have demonstrated selectivity by virtueof their ability to target the more poorly conserved areas of theATP-binding site.

One of the principal mechanisms by which cellular regulation is effectedis through the transduction of extracellular signals across the membranethat in turn modulate biochemical pathways within the cell. Proteinphosphorylation represents one course by which intracellular signals arepropagated from molecule to molecule resulting finally in a cellularresponse. These signal transduction cascades are highly regulated andoften overlapping as evidenced by the existence of many protein kinasesas well as phosphatases. It is currently believed that a number ofdisease states and/or disorders are a result of either aberrantactivation or functional mutations in the molecular components of kinasecascades.

MAPKAP-K2 is a serine/threonine kinase that operates immediatelydownstream of the p38 within the stress-induced MAPK pathway (FIG. 1).

The p38 pathway is involved in transducing the effects of a variety ofstress-related extracellular stimuli such as heat shock, UV light,bacterial lipopolysaccharide, and pro-inflammatory cytokines. Activationof this pathway results in the phosphorylation of transcription andinitiation factors, and affects cell division, apoptosis, invasivenessof cultured cells and the inflammatory response (Martin-Blanco,Bioessays 22, 637-645 (2000)).

p38 itself activates a number of protein kinases other than the MAPKAPkinases such as Mnk1/2, PRAK and MSK1 (FIG. 1). The specific and/oroverlapping functions of the majority of these downstream targets haveyet to be resolved. This pathway has been of particular interest for thediscovery of new anti-inflammatory agents. Previous strategies tointervene in this pathway have involved the development of selectiveinhibitors of p38 itself. Such inhibitors have proven efficacy ininhibiting pro-inflammatory cytokine production in cell-based models anddemonstrated efficacy in animal models of chronic inflammatoryconditions (Lee et al., Immunopharmacology 47, 185-201 (2000)). However,knockout of p38 expression in mouse models results in embryoniclethality, furthermore cells derived from such embryos have demonstrateda number of effects on fundamental cell responses. These observationsindicate that caution should be applied to therapies involving thelong-term dosing of humans with p38 inhibitors.

An alternative strategy for the development of anti-inflammatory agentscould be the inhibition of this pathway at the level of MAPKAP-K2. HumanMAPKAP-K2 has two proline-rich segments at its N-terminus followed bythe kinase domain and the C-terminal regulatory domain. The kinase haslow homology with other serine/threonine kinases except MAPKAP-K3 and 4.The C-terminal regulatory domain contains a bipartite nuclearlocalisation signal and a nuclear export signal. The crystal structureof inactive MAPKAP-K2 has been resolved (Meng, W. et al. J. Biol. Chem.277, 37401-37405 (2002)). Activation of MAPKAP-K2 by p38 occurs via theselective phosphorylation of threonine residues 222 and 334 (Stokoe etal., EMBO J. 11, 3985-3994 (1992)). MAPKAP-K2 has an amphiphilic A-helixmotif located within its C-terminal region that is likely to act toblock the binding of substrates. The dual phosphorylation by p38 hasbeen proposed to reposition this motif resulting in enhanced catalyticactivity (You-Li et al., J. Biol. Chem. 270, 202-206 (1995)). MAPKAP-K2is present in the nucleus of unstimulated cells and translocates to thecytoplasm upon cell activation. This kinase is known to phosphorylate anumber of nuclear transcription factors as well as cytosolic proteinssuch as the heat shock proteins and 5-lipoxygenase (Stokoe et al., FEBSLett. 313, 307-313 (1992), Werz, et al., Proc. Natl. Acad. Sci. USA 97,5261-5266 (2000), Heidenreich, et al., J. Biol. Chem. 274, 14434-14443(1999), Tan, et al., EMBO J. 15, 4629-4642 (1996), Neufeld, J. Biol.Chem. 275, 20239-20242 (2000)). All such substrates contain a uniqueamino acid motif (XX-Hyd-XRXXSXX, where Hyd is a bulky hydrophobicresidue) that is required for efficient phosphorylation by MAPKAP-K2(Stokoe et al., Biochem. J. 296, 843-849 (1993)).

Currently MAPKAP-K2 is the only p38 substrate for which a specificfunction has been identified. A specific role for MAPKAP-K2 in mediatingthe inflammatory response has been strongly indicated by the phenotypeof the MAPKAP-K2-deficient mouse (MAPKAP-K2^(−/−)) (Kotlyarov, et al.,Nature Cell Biol. 1, 94-97 (1999)). This mouse is viable and normalexcept for a significantly reduced inflammatory response. Recently ithas also been shown that MAPKAP-K2 deficiency results in a markedneuroprotection from ischaemic brain injury (Wang et al., J. Biol. Chem.277, 43968-43972 (2002)). MAPKAP-K2 is believed to regulate thetranslation and/or stability of important pro-inflammatory cytokinemRNAs. It is thought to perform this function via the phosphorylation ofproteins that bind to the AU-rich elements found within untranslatedregions of these cytokines. The identity of these proteins is currentlyunder investigation.

MAPKAP-K2 therefore represents a targeted intervention point in thestress-induced kinase cascade for perturbation of the inflammatoryresponse.

There exists a need for the provision of compounds that are inhibitorsof MAPKAP-K2 kinases.

DISCLOSURE OF THE INVENTION

As a result of much diligent research directed toward achieving theobject stated above, the present inventors have completed the presentinvention upon discovering that the Pyrazolo[1,5-a]pyrimidinederivatives represented by formula (I) below and their pharmaceuticallyacceptable salts exhibit excellent kinase inhibiting activity.

In other words, the present invention provides as follows:(1) A use of a compound of formula (I):

wherein R¹ is hydrogenR² is hydrogenR³ is C1-C8 optionally substituted alkyl, C2-C8 optionally substitutedalkenyl, C2-C8 optionally substituted alkynyl, C3-C8 optionallysubstituted cycloalkyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted arylalkyl, optionallysubstituted heteroarylalkyl, optionally substituted arylalkenyl,optionally substituted heteroarylalkenyl, optionally substitutedarylalkynyl, or optionally substituted heteroarylalkynyl;R⁴ is hydrogen;R⁵ is C1-C8 optionally substituted alkyl, C2-C8 optionally substitutedalkenyl, C2-C8 optionally substituted alkynyl, C3-C8 optionallysubstituted cycloalkyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted arylalkyl, optionallysubstituted heteroarylalkyl, optionally substituted arylalkenyl,optionally substituted heteroarylalkenyl, optionally substitutedarylalkynyl, or optionally substituted heteroarylalkynyl, optionallysubstituted heterocyclyl or optionally substituted heterocyclylalkyl;R⁶ is hydrogen, C1-C8 optionally substituted alkyl, C2-C8 optionallysubstituted alkenyl, C2-C8 optionally substituted alkynyl or C3-C8optionally substituted cycloalkyl;or R⁵ and R⁶ together may be taken together with the nitrogen to whichthey are attached to form a mono or bicyclic heterocycle with 5-7members in each ring and optionally containing, in addition to thenitrogen, one or two additional heteroatoms selected from N, O and S,the said mono or bicyclic heterocycle may optionally be substituted withone or more substituents;or pharmaceutically acceptable salts, or other pharmaceuticallyacceptable biohydrolyzable derivatives thereof, including esters,amides, carbamates, carbonates, ureides, solvates, hydrates, affinityreagents or prodrugs thereof, in the manufacture of a medicament for usein inhibiting protein kinases.(2) The use as (1), wherein R³ is C1-C8 optionally substituted alkyl,C2-C8 optionally substituted alkenyl, C2-C8 optionally substitutedalkynyl, C3-C8 optionally substituted cycloalkyl, optionally substitutedaryl, optionally substituted heteroaryl, optionally substitutedarylalkyl or optionally substituted heteroarylalkyl.(3) The use as (2), wherein R³ is C2-C8 optionally substituted alkenyl,optionally substituted aryl or optionally substituted arylalkyl.(4) The use as any one of (1) to (3), wherein R⁵ is C1-C8 optionallysubstituted alkyl, C2-C8 optionally substituted alkenyl, C2-C8optionally substituted alkynyl, C3-C8 optionally substituted cycloalkyl,optionally substituted heterocyclyl or optionally substitutedheterocyclylalkyl.(5) The use as (4), wherein R⁵ is C3-C8 cycloalkyl substituted by NHR⁷,where R⁷ is optionally substituted heterocyclyl or optionallysubstituted heterocyclylalkyl.(6) The use as any one of (1) to (5), wherein R⁶ is hydrogen or C1-C8optionally substituted alkyl.(7) The use as (6), wherein R⁶ is hydrogen.(8) The use as any one of (1) to (7), wherein the medicament is for useas an inhibitor of MAPKAP-K2.(9) The use as (8), wherein the medicament is for use in the preventionor treatment of a MAPKAP-K2-mediated disorder.(10) The use as (9), wherein the MAPKAP-K2 mediated disorder is aneurological disorder (including dementia), an inflammatory disease, adisorder linked to apoptosis, particularly neuronal apoptosis, stroke,sepsis, autoimmune disease, destructive bone disorder, proliferativedisorder, cancer, infectious disease, allergy, ischemia reperfusioninjury, heart attack, angiogenic disorder, organ hypoxia, vascularhyperplasia, cardiac hypertrophy, thrombin induced platelet aggregation.(11) The use as (10), wherein the disorder is a neurodegenerativedisorder.(12) The use as (11), wherein the neurodegenerative disorder isdementia, Alzheimer's disease, Parkinson's disease, Amyotrophic LateralSclerosis, Huntington's disease, senile chorea, Sydenham's chorea,hypoglycemia, head and spinal cord trauma including traumatic headinjury, acute and chronic pain, epilepsy and seizures,olivopontocerebellar dementia, neuronal cell death, hypoxia-relatedneurodegeneration, acute hypoxia, glutamate toxicity including glutamateneurotoxicity, cerebral ischemia, dementia linked to meningitis and/orneurosis, cerebrovascular dementia, or dementia in an HIV-infectedpatient.(13) The use as (10), wherein the disorder results from inflammation.(14) The use as (13), wherein the disorder is inflammatory boweldisorder, bronchitis, asthma, acute pancreatitis, chronic pancreatitis,allergies of various types or Alzheimer's disease.(15) The use as (10), wherein the disorder is an autoimmune disease.(16) The use as (15), wherein the autoimmune disease is rheumatoidarthritis, systemic lupus erythematosus, glomerulonephritis,scleroderma, chronic thyroiditis, Graves's disease, autoimmunegastritis, diabetes, autoimmune haemolytis anaemia, autoimmuneneutropaenia, thrombocytopenia, atopic dermatitis, chronic activehepatitis, myasthenia gravis, multiple sclerosis, ulcerative colitis,Crohn's disease, psoriasis or graft vs host disease.(17) A method of treating or preventing a MAPKAP-K2-mediated disorder inan individual, which comprises administering to said individual at leastone compound as defined in any one of (1) to (7) or a compositiondefined in (8) or (9).(18) The method as (17), wherein the MAPKAP-K2 mediated disorder is aneurological disorder (including dementia), an inflammatory disease, adisorder linked to apoptosis, particularly neuronal apoptosis, stroke,sepsis, autoimmune disease, destructive bone disorder, proliferativedisorder, cancer, infectious disease, allergy, ischemia reperfusioninjury, heart attack, angiogenic disorder, organ hypoxia, vascularhyperplasia, cardiac hypertrophy, thrombin induced platelet aggregation.(19) The method as (18), wherein the disorder is a neurodegenerativedisorder.(20) The method as (19), wherein the neurodegenerative disorder isdementia, Alzheimer's disease, Parkinson's disease, Amyotrophic LateralSclerosis, Huntington's disease, senile chorea, Sydenham's chorea,hypoglycemia, head and spinal cord trauma including traumatic headinjury, acute and chronic pain, epilepsy and seizures,olivopontocerebellar dementia, neuronal cell death, hypoxia-relatedneurodegeneration, acute hypoxia, glutamate toxicity including glutamateneurotoxicity, cerebral ischemia, dementia linked to meningitis and/orneurosis, cerebrovascular dementia, or dementia in an HIV-infectedpatient.(21) The method as (18), wherein the disorder results from inflammation.(22) The method as (21), wherein the disorder is inflammatory boweldisorder, bronchitis, asthma, acute pancreatitis, chronic pancreatitis,allergies of various types or Alzheimer's disease.(23) The method as (18), wherein the disorder is an autoimmune disease.(24) The method as (23), wherein the autoimmune disease is rheumatoidarthritis, systemic lupus erythematosus, glomerulonephritis,scleroderma, chronic thyroiditis, Graves's disease, autoimmunegastritis, diabetes, autoimmune haemolytis anaemia, autoimmuneneutropaenia, thrombocytopenia, atopic dermatitis, chronic activehepatitis, myasthenia gravis, multiple sclerosis, ulcerative colitis,Crohn's disease, psoriasis or graft vs host disease.(25) The method as any one of (18) to (24), wherein one or more activeagents is/are administered to the individual simultaneously,subsequently or sequentially to administering the compound.(26) The method for determining the activity of the compounds as definedin any one of (1) to (7), comprising providing a system for assaying theactivity and assaying the activity of a compound as defined in any of(1) to (7).(27) The method as (26), wherein the assay is for the protein kinaseinhibiting activity of the compound.(28) A method of inhibiting the activity or function of a proteinkinase, which comprises exposing a protein kinase to the compound asdefined in any of (1) to (7).(29) A method of inhibiting the activity or function of MAPKAP-K2, whichcomprises exposing MAPKAP-K2 to the compound as defined in any of (1) to(7).(30) The method as (29) which is performed in a research model, invitro, in silico, or in vivo such as in an animal model.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the cascade of the p38 within the stress-induced MAPKpathway.

FIG. 2 shows a general reaction scheme for the preparation of compoundsof Formula I. The invention will now be illustrated by the followingnon-limiting examples.

BEST MODE FOR CARRYING OUT THE INVENTION

For the purposes of this invention, alkyl relates to both straight chainand branched alkyl radicals of 1 to 8 carbon atoms including but notlimited to methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,isobutyl, tert-butyl n-pentyl and n-hexyl. The term also encompassescycloalkyl radicals of C3 to C8 carbon atoms including but not limitedto cyclopropyl, cyclobutyl, CH₂-cyclopropyl, CH₂-cyclobutyl, cyclopentylor cyclohexyl.

The term “alkenyl” means a straight chain or branched alkenyl radical of2 to 8 carbon atoms and containing one or more carbon-carbon doublebonds and includes but is not limited to ethylene, n-propyl-1-ene,n-propyl-2-ene, isopropylene, etc.

The term “alkynyl” means a straight chain or branched alkynyl radical of2 to 8 carbon atoms and containing one or more carbon-carbon triplebonds and includes but is not limited to ethynyl, 2-methylethynyl etc.

“Aryl” means an aromatic 3-10 membered hydrocarbon containing one ringor being fused to one or more saturated or unsaturated rings includingbut not limited to phenyl, naphthyl, anthracenyl or phenanthracenyl.

“Heteroaryl” means an aromatic 3-10 membered aryl containing one or moreheteroatoms selected from N, O or S and containing one ring or beingfused to one or more saturated or unsaturated rings and;

“Heterocyclyl” means a 3-10 membered ring system containing one or moreheteroatoms selected from N, O or S and includes heteroaryl. Theheterocyclyl system can contain one ring or may be fused to one or moresaturated or unsaturated rings; the heterocyclyl can be fully saturated,partially saturated or unsaturated and includes but is not limited toheteroaryl and heterocarbocyclyl. Examples of carbocyclyl orheterocyclyl groups include but are not limited to cyclohexyl, phenyl,acridine, benzimidazole, benzofuran, benzothiophene, benzoxazole,benzothiazole, carbazole, cinnoline, dioxin, dioxane, dioxolane,dithiane, dithiazine, dithiazole, dithiolane, furan, imidazole,imidazoline, imidazolidine, indole, indoline, indolizine, indazole,isoindole, isoquinoline, isoxazole, isothiazole, morpholine,napthyridine, oxazole, oxadiazole, oxathiazole, oxathiazolidine,oxazine, oxadiazine, phenazine, phenothiazine, phenoxazine, phthalazine,piperazine, piperidine, pteridine, purine, pyran, pyrazine, pyrazole,pyrazoline, pyrazolidine, pyridazine, pyridine, pyrimidine, pyrrole,pyrrolidine, pyrroline, quinoline, quinoxaline, quinazoline,quinolizine, tetrahydrofuran, tetrazine, tetrazole, thiophene,thiadiazine, thiadiazole, thiatriazole, thiazine, thiazole,thiomorpholine, thianaphthalene, thiopyran, triazine, triazole, andtrithiane.

Halogen means F, Cl, Br or I.

Suitable substituents include alkyl, cycloalkyl, heterocyclyl, alkenyl,alkynyl, alkoxy, aryloxy, halogen, hydroxy, NO₂, CN, CO₂R¹⁴, CONR¹⁴R¹⁵,NR¹⁴(CO)_(n)R¹⁵, S(O)_(m)R¹⁴; where R¹⁴ and R¹⁵, which may be the sameor different, are hydrogen, alkyl or aryl; n is 0.1; m is 0 μl or 2.

Preferably, R³ is C1-C8 optionally substituted alkyl, C2-C8 optionallysubstituted alkenyl, C2-C8 optionally substituted alkynyl, C3-C8optionally substituted cycloalkyl, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted arylalkyl oroptionally substituted heteroarylalkyl;

More preferably R³ is C1-C8 optionally substituted alkenyl, optionallysubstituted aryl or optionally substituted arylalkyl

Preferably, R⁵ is C1-C8 optionally substituted alkyl, C2-C8 optionallysubstituted alkenyl, C2-C8 optionally substituted alkynyl, C3-C8optionally substituted cycloalkyl, optionally substituted heterocyclylor optionally substituted heterocyclylalkyl.

More preferably R⁵ is C3-C8 cycloalkyl substituted by NHR⁷, where R⁷ isoptionally substituted heterocyclyl or optionally substitutedheterocyclylalkyl.

Preferably R⁶ is hydrogen or C1-C8 optionally substituted alkyl. Morepreferably R⁶ is hydrogen.

As preferred combinations of the groups mentioned as preferred examplesof R¹-R⁶ in formula I according to the invention, there may be mentionedthe following combinations 1) or 2).

-   1) In formula I, wherein R¹ is hydrogen, R² is hydrogen, R³ is    C6-C14 optionally substituted aryl, R⁴ is hydrogen, R⁵ is C3-C8    optionally substituted cycloalkyl and R⁶ is hydrogen.-   2) In formula I, wherein R¹ is hydrogen, R² is hydrogen, R³ is    C6-C14 optionally substituted aryl, R⁴ is hydrogen, R⁵ is optionally    substituted heterocyclyl and R⁶ is hydrogen.

The compounds for use in the first aspect may be provided as a salt,preferably as a pharmaceutically acceptable salt of compounds of formulaI. Examples of pharmaceutically acceptable salts of these compoundsinclude those derived from organic acids such as acetic acid, malicacid, tartaric acid, citric acid, lactic acid, oxalic acid, succinicacid, fumaric acid, maleic acid, benzoic acid, salicylic acid,phenylacetic acid, mandelic acid, methanesulphonic acid,benzenesulphonic acid and p-toluenesulphonic acid, mineral acids such ashydrochloric and sulphuric acid and the like, giving methanesulphonate,benzenesulphonate, p-toluenesulphonate, hydrochloride and sulphate, andthe like, respectively or those derived from bases such as organic andinorganic bases. Examples of suitable inorganic bases for the formationof salts of compounds for this invention include the hydroxides,carbonates, and bicarbonates of ammonia, lithium, sodium, calcium,potassium, aluminium, iron, magnesium, zinc and the like. Salts can alsobe formed with suitable organic bases. Such bases suitable for theformation of pharmaceutically acceptable base addition salts withcompounds of the present invention include organic bases which arenontoxic and strong enough to form salts. Such organic bases are alreadywell known in the art and may include amino acids such as arginine andlysine, mono-, di-, or trihydroxyalkylamines such as mono-, di-, andtriethanolamine, choline, mono-, di-, and trialkylamines, such asmethylamine, dimethylamine, and trimethylamine, guanidine;N-methylglucosamine; N-methylpiperazine; morpholine; ethylenediamine;N-benzylphenethylamine; tris(hydroxymethyl)aminomethane; and the like.

Salts may be prepared in a conventional manner using methods well knownin the art. Acid addition salts of said basic compounds may be preparedby dissolving the free base compounds according to the first or secondaspects of the invention in aqueous or aqueous alcohol solution or othersuitable solvents containing the required acid. Where a compound of theinvention contains an acidic function, a base salt of said compound maybe prepared by reacting said compound with a suitable base. The acid orbase salt may separate directly or can be obtained by concentrating thesolution e.g. by evaporation. The compounds of this invention may alsoexist in solvated or hydrated forms.

The invention also extends to the use of a prodrug of the aforementionedcompounds such as an ester or amide thereof. A prodrug is any compoundthat may be converted under physiological conditions or by solvolysis toany of the compounds of the invention or to a pharmaceuticallyacceptable salt of the compounds of the invention. A prodrug may beinactive when administered to a subject but is converted in vivo to anactive compound of the invention.

The compounds for use according to the invention may contain one or moreasymmetric carbon atoms and may exist in racemic and optically activeforms. The compounds of the invention may exist in trans or cis form.The first aspect of the invention covers the use of all such compounds.

As specific examples of compounds of the formula I above there may bementioned compounds listed in Table A below.

Wherein “Me” and “Ph” mean “methyl group” and “phenyl group”respectively. TABLE A Com- pound No. R¹ R² R⁴ R³ NR⁵R⁶  1 H H H

 2 H H H

 3 H H H

 4 H H H

 5 H H H

 6 H H H

 7 H H H

 8 H H H

 9 H H H

 10 H H H

 11 H H H

 12 H H H

 13 H H H

 14 H H H

 15 H H H

 16 H H H

 17 H H H

 18 H H H

 19 H H H

 20 H H H

 21 H H H

 22 H H H

 23 H H H

 24 H H H

 25 H H H

 26 H H H

 27 H H H

 28 H H H

 29 H H H

 30 H H H

 31 H H H

 32 H H H

 33 H H H

 34 H H H

 35 H H H

 36 H H H

 37 H H H

 38 H H H

 39 H H H

 40 H H H

 41 H H H

 42 H H H

 43 H H H

 44 H H H

 45 H H H

 46 H H H

 47 H H H

 48 H H H

 49 H H H

 50 H H H

 51 H H H

 52 H H H

 53 H H H

 54 H H H

 55 H H H

 56 H H H

 57 H H H

 58 H H H

 59 H H H

 60 H H H

 61 H H H

 62 H H H

 63 H H H

 64 H H H

 65 H H H

 66 H H H

 67 H H H

 68 H H H

 69 H H H

 70 H H H

 71 H H H

 72 H H H

 73 H H H

 74 H H H

 75 H H H

 76 H H H

 77 H H H

 78 H H H

 79 H H H

 80 H H H

 81 H H H

 82 H H H

 83 H H H

 84 H H H

 85 H H H

 86 H H H

 87 H H H

 88 H H H

 89 H H H

 90 H H H

 91 H H H

 92 H H H

 93 H H H

 94 H H H

 95 H H H

 96 H H H

 97 H H H

 98 H H H

 99 H H H

100 H H H

101 H H H

102 H H H

103 H H H

104 H H H

105 H H H

106 H H H

107 H H H

108 H H H

109 H H H

110 H H H

111 H H H

112 H H H

113 H H H

114 H H H

115 H H H

116 H H H

117 H H H

118 H H H

119 H H H

120 H H H

121 H H H

122 H H H

123 H H H

124 H H H

125 H H H

126 H H H

127 H H H

128 H H H

129 H H H

130 H H H

131 H H H

132 H H H

133 H H H

134 H H H

135 H H H

136 H H H

137 H H H

138 H H H

139 H H H

140 H H H

141 H H H

142 H H H

143 H H H

144 H H H

145 H H H

146 H H H

147 H H H

148 H H H

149 H H H

150 H H H

151 H H H

152 H H H

153 H H H

154 H H H

155 H H H

156 H H H

157 H H H

158 H H H

159 H H H

160 H H H

161 H H H

162 H H H

163 H H H

164 H H H

165 H H H

166 H H H

167 H H H

168 H H H

169 H H H

170 H H H

171 H H H

172 H H H

173 H H H

174 H H H

175 H H H

176 H H H

177 H H H

178 H H H

179 H H H

180 H H H

181 H H H

182 H H H

183 H H H

184 H H H

185 H H H

186 H H H

187 H H H

188 H H H

189 H H H

190 H H H

191 H H H

192 H H H

193 H H H

194 H H H

195 H H H

196 H H H

197 H H H

198 H H H

199 H H H

200 H H H

201 H H H

202 H H H

203 H H H

204 H H H

205 H H H

206 H H H

207 H H H

208 H H H

209 H H H

210 H H H

211 H H H

212 H H H

213 H H H

214 H H H

215 H H H

216 H H H

217 H H H

218 H H H

219 H H H

220 H H H

221 H H H

222 H H H

223 H H H

224 H H H

225 H H H

226 H H H

227 H H H

228 H H H

229 H H H

Suitably, the compounds as defined herein are inhibitors of MAPKAP-K2.For the purpose of this invention, an inhibitor is any compound whichreduces or prevents the activity of the MAPKAP-K2 enzyme.

A “MAPKAP-K2-mediated disorder” is any disease or deleterious conditionin which MAPKAP-K2 plays a role. Examples include neurological disorder(including dementia), inflammatory disease, a disorder linked toapoptosis, particularly neuronal apoptosis, stroke, sepsis, autoimmunedisease, destructive bone disorder, proliferative disorder, cancer,infectious disease, allergy, ischemia reperfusion injury, heart attack,angiogenic disorder, organ hypoxia, vascular hyperplasia, cardiachypertrophy, thrombin induced platelet aggregation.

The compounds as defined herein are particularly useful for theprevention or treatment of a neurodegenerative disorder. In particular,the neurodegenerative disorder results from apoptosis and/orinflammation. Examples of neurodegenerative disorders are: dementia;Alzheimer's disease; Parkinson's disease; Amyotrophic Lateral Sclerosis;Huntington's disease; senile chorea; Sydenham's chorea; hypoglycemia;head and spinal cord trauma including traumatic head injury; acute andchronic pain; epilepsy and seizures; olivopontocerebellar dementia;neuronal cell death; hypoxia-related neurodegeneration; acute hypoxia;glutamate toxicity including glutamate neurotoxicity; cerebral ischemia;dementia linked to meningitis and/or neurosis; cerebrovascular dementia;or dementia in an HIV-infected patient.

The compounds as defined herein can also be used to prevent or treatdisorders resulting from inflammation. These include, for example,inflammatory bowel disorder, bronchitis, asthma, acute pancreatitis,chronic pancreatitis, allergies of various types, and possiblyAlzheimer's disease. Autoimmune diseases which may also be treated orprevented by the compounds of the present invention include rheumatoidarthritis, systemic lupus erythematosus, glomerulonephritis,scleroderma, chronic thyroiditis, Graves's disease, autoimmunegastritis, diabetes, autoiminune haemolytis anaemia, autoimmuneneutropaenia, thrombocytopenia, atopic dermatitis, chronic activehepatitis, myasthenia gravis, multiple sclerosis, ulcerative colitis,Crohn's disease, psoriasis or graft vs host disease.

Compounds for use according to the present invention can be prepared asfollows: by reaction of a compound of formula II, III, or VI as follows,wherein R¹-R⁶ are as defined above:1) reacting a compound of the formula II

with a compound of the formula R⁵R⁶NH either in the absence or presenceof metal catalysis under e.g. Buchwald conditions (J. Am. Chem. Soc.116, 7901-7902 (1994)), and removal of the protecting group with forexample CF₃CO₂H (for example as described in Protective Groups inOrganic Synthesis, 3rd Ed, John Wiley & Sons Inc)2) reacting a compound of the formula III

with a compound of the formula R⁵R⁶NH3) reacting a compound of the formula III

with a compound of the formula ((CH₃)₃COCO)₂O (for example as describedin Protective Groups in Organic Synthesis, 3rd Ed, John Wiley & SonsInc)4) reacting a compound of the formula IV

with a compound of the formula R³NH₂ or R³NHAc

A compound of formula I may undergo one or more further reactions toprovide a different compound of formula I. For example, a compound mayundergo a reduction, oxidation, elimination, substitution and/oraddition reaction.

The compounds of formula IV are either known or can be prepared bymethods analogous to those known for preparing analogous knowncompounds. Compounds of formula II and III include novel compounds andsuch novel compounds form an additional aspect of the invention.

Other methods will be apparent to the chemist skilled in the art, aswill the methods for preparing starting materials and intermediates. TheExamples also make apparent various methods of preparing compounds ofthe invention as well as starting materials and intermediates.

Medicaments as defined herein may also comprise one or more additionalactive agents, such as an anti-inflammatory agent (for example a p38inhibitor, glutamate receptor antagonist, or a calcium channelantagonist), a chemotherapeutic agent and/or an antiproliferative agent.

Suitable carriers and/or diluents are well known in the art and includepharmaceutical grade starch, mannitol, lactose, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, (or other sugar),magnesium carbonate, gelatin, oil, alcohol, detergents, emulsifiers orwater (preferably sterile). The composition may be a mixed preparationof a composition or may be a combined preparation for simultaneous,separate or sequential use (including administration).

The medicaments may be administered by any convenient method, forexample by oral (including by inhalation), parenteral, mucosal (e.g.buccal, sublingual, nasal), rectal or transdermal administration and thecompositions adapted accordingly.

For oral administration, the composition can be formulated as liquids orsolids, for example solutions, syrups, suspensions or emulsions,tablets, capsules and lozenges.

A liquid formulation will generally consist of a suspension or solutionof the compound or physiologically acceptable salt in a suitable aqueousor non-aqueous liquid carrier(s) for example water, ethanol, glycerine,polyethylene glycol or an oil. The formulation may also contain asuspending agent, preservative, flavouring or colouring agent.

A composition in the form of a tablet can be prepared using any suitablepharmaceutical carrier(s) routinely used for preparing solidformulations. Examples of such carriers include magnesium stearate,starch, lactose, sucrose and microcrystalline cellulose.

A composition in the form of a capsule can be prepared using routineencapsulation procedures. For example, powders, granules or pelletscontaining the active ingredient can be prepared using standard carriersand then filled into a hard gelatin capsule; alternatively, a dispersionor suspension can be prepared using any suitable pharmaceuticalcarrier(s), for example aqueous gums, celluloses, silicates or oils andthe dispersion or suspension then filled into a soft gelatin capsule.

Compositions for oral administration may be designed to protect theactive ingredient against degradation as it passes through thealimentary tract, for example by an outer coating of the formulation ona tablet or capsule.

Typical parenteral compositions consist of a solution or suspension ofthe compound or physiologically acceptable salt in a sterile aqueous ornon-aqueous carrier or parenterally acceptable oil, for examplepolyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil orsesame oil. Alternatively, the solution can be lyophilised and thenreconstituted with a suitable solvent just prior to administration.

Compositions for nasal or oral administration may conveniently beformulated as aerosols, drops, gels and powders. Aerosol formulationstypically comprise a solution or fine suspension of the active substancein a physiologically acceptable aqueous or non-aqueous solvent and areusually presented in single or multidose quantities in sterile form in asealed container, which can take the form of a cartridge or refill foruse with an atomising device. Alternatively the sealed container may bea unitary dispensing device such as a single dose nasal inhaler or anaerosol dispenser fitted with a metering valve which is intended fordisposal once the contents of the container have been exhausted. Wherethe dosage form comprises an aerosol dispenser, it will contain apharmaceutically acceptable propellant. The aerosol dosage forms canalso take the form of a pump-atomiser.

Compositions suitable for buccal or sublingual administration includetablets, lozenges and pastilles, wherein the active ingredient isformulated with a carrier such as sugar and acacia, tragacanth, orgelatin and glycerin.

Compositions for rectal or vaginal administration are conveniently inthe form of suppositories (containing a conventional suppository basesuch as cocoa butter), pessaries, vaginal tabs, foams or enemas.

Compositions suitable for transdermal administration include ointments,gels, patches and injections including powder injections.

Conveniently the composition is in unit dose form such as a tablet,capsule or ampoule.

Manufacture of the medicaments can be carried out by standard techniqueswell known in the art. The composition may be in any form including atablet, a liquid, a capsule, and a powder or in the form of a foodproduct, e.g. a functional food. In the latter case the food productitself may act as the pharmaceutically acceptable carrier.

A compound as defined herein may be administered simultaneously,subsequently or sequentially with one or more other active agent, suchas an anti-inflammatory agent e.g. p38 inhibitor, glutamate receptorantagonist, calcium channel antagonist, a chemotherapeutic agent or anantiproliferative agent. For example, for acute treatment, a p38inhibitor may be administered to a patient prior to administering acompound of the present invention.

The compounds as defined herein will normally be administered in a dailydosage regimen (for an adult patient) of, for example, an oral dose ofbetween 1 mg and 2000 mg, preferably between 30 mg and 1000 mg, e.g.between 10 and 250 mg or an intravenous, subcutaneous, or intramusculardose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 50 mg,e.g. between 1 and 25 mg of the compound of the formula (I) or aphysiologically acceptable salt thereof calculated as the free base, thecompound being administered 1 to 4 times per day. Suitably the compoundswill be administered for a period of continuous therapy, for example fora week or more.

In a second aspect, the present invention provides a method of treatingor preventing a MAPKAP-K2-mediated disorder in an individual, whichcomprises administering to said individual a compound as defined herein.The active compound is preferably administered in a cumulative effectiveamount. The individual may be in need of the treatment or prevention.Any of the MAPKAP-K2-mediated disorders discussed above may be thesubject of treatment or prevention. One or more other active agents maybe administered to the individual simultaneously, subsequently orsequentially to administering the compound. The other active agent maybe an anti-inflammatory agent such as a p38 inhibitor, glutamatereceptor antagonist, calcium channel antagonist, a chemotherapeuticagent or an antiproliferative agent.

In a third aspect, the present invention provides an assay fordetermining the activity of the compounds as defined herein, comprisingproviding a system for assaying the activity and assaying the activityof the compound. Preferably the assay is for the MAPKAP-K2 inhibitingactivity of the compound. The compounds as defined herein may be assayedin vitro, in vivo, in silico, or in a primary cell culture or a cellline. In vitro assays include assays that determine inhibition of thekinase activity of activated MAPKAP-K2. Alternatively, in vitro assaysmay quantitate the ability of a compound to bind MAPKAP-K2 and may bemeasured either by radiolabelling the compound prior to binding, thenisolating the inhibitor/MAPKAP-K2 complex and determining the amount ofthe radiolabel bound or by running a competition experiment where newinhibitors are incubated with MAPKAP-K2 bound to known radioligands. Anexample of an assay, which may be used, is Scintillation Proximity Assay(SPA), preferably using radiolabelled ATP. Another example is ELISA. Anytype or isoform of MAPKAP-K2 may be used in these assays.

In an fourth aspect, the present invention provides a method ofinhibiting the activity or function of a MAPKAP-12, which comprisesexposing a MAPKAP-K2 to a compound or a composition of the first orfourth aspect of the present invention. The method may be performed in aresearch model, in vitro, in silico, or in vivo such as in an animalmodel. A suitable animal model may be a kainic acid model in rat ormice, traumatic brain injury model in rat, or MPTP in mice.

All features of each of the aspects apply to all other aspects mutatismutandis.

EXAMPLES

The invention will now be explained in greater detail by the followingexamples, with the understanding that the scope of the invention is notin any sense restricted by these examples.

Example 1

[General Procedures for the Synthesis of Pyrazolo[1,5-a]pyrimidines ofGeneral Formula (III)]

a) To a solution of 5,7-dichloropyrazolo[1,5-a]pyrimidine (IV) (2 g) andtriethylamine (2 equivalents) in 2-propanol (20 ml) was added the amineR³NH₂ (1 or 1.1 equivalents) and the mixture was stirred at roomtemperature overnight. The mixture was concentrated in vacuo and theresidue was then partitioned between water and dichloromethane. Theorganic phase was washed twice with water and the combined aqueousphases back-extracted with dichloromethane. The combined organic layerswere combined, washed with brine and dried over Na₂SO₄. Removal of thesolvent in vacuo yielded the precursor (III). (Purificationperformed—normally the products did not require any furtherpurification, if they did, they were recrystallised. Analysisperformed—NMR, HPLC and MS.)

Should the above room-temperature reaction not occur satisfactorily, thefollowing may be applied:

b) To a solution of 5,7-dichloropyrazolo[1,5-a]pyrimidine (IV) (2 g) in2-propanol (25 ml) containing N,N-diisopropylethylamine (2 equivalents)was added the amine R³NH₂ (1.2 equivalents). The reaction was heatedovernight at 80° C. and the solvent removed in vacuo. The residue waspartitioned between water and dichloromethane and the organic phase waswashed with water, brine and dried over MgSO₄. Removal of the solvent invacuo yielded the product.

In those cases where R³NH₂ is a hindered or weakly nucleophilic anilinethe following procedure may be applied:

c) To a solution of 2-methylacetanilide (2.2 mmol) in toluene (3 ml) atroom temperature was added sodium hydride (3 mmol) after the additionthe mixture was heated until effervescence ceased and the solutionbecame homogenous. 5,7-Dichloropyrazolo[1,5-a]pyrimidine (IV) (1 mmol)was added and the mixture heated at reflux for 5 h. (The solutionbecomes heterogeneous during this time). Upon cooling, acetic acid (1ml) and water (1 ml) were cautiously added and the mixture was stirredfor 15 min. The solvent was removed in vacuo and the residual aceticacid removed by azeotropic evaporation with toluene, (3×). The residuewas partitioned between water and ethyl acetate. The organic phase waswashed (water and brine) and dried. The solvent was removed in vacuo andthe residue was chromatographed to afford the desired compound (III).Typical unoptimised yields for c) 50-70%. The Rf of starting material(IV) and product (III) are chromatographically indistinguishable, makingcomplete reaction difficult to determine. It appears that at least 5 his required for significant reaction to occur. Compound No R¹ R² R⁴ R³Mp (° C.) IIIA H H H 2-Me 119-121 phenyl IIIB H H H 2,4-Cl₂ 120-128phenyl

Example 2

[General Procedure for the Synthesis of Pyrazolo[1,5-a]pyrimidines ofGeneral Formula (II)]

To a solution of the precursor (II) formed above (2 g) in 1,4-dioxane(10 ml) was added di-tert-butyl dicarbonate (2 equivalents) in1,4-dioxane (10 ml) followed by 4-dimethylaminopyridine (cat). Thereaction was stirred at room temperature overnight and if startingmaterial was detected by TLC, the reaction was left for longer. Themixture was concentrated in vacuo and the residue was then partitionedbetween water and dichloromethane. The organic phase washed with 10%citric acid, water and brine and then dried over MgSO₄. Removal of thesolvent it vacuo gave the Boc protected intermediate (II). (Purificationperformed—filter column to remove any residual 4-dimethylaminopyridine.Analysis performed—NMR, HPLC and MS.) Compound No R¹ R² R⁴ R³ ¹HNMR(CDCl₃) IIA H H H 2-Me 1.38(9H, s, tBu), phenyl 2.3(3H, s, CH₃),6.4(1H, s, Het-H), 6.44(1H, s, Het-H), 7.15-7.34(4H, m, ArH), 8.15(1H,s, Het-H) IIC H H H 2-F phenyl 1.4(9H, s, tBu), 6.67(1H, m, 2Het-H),7.08-7.4(4H, m, ArH) 8.17(1H, s, Het-H)

Example 3

[General Procedures for the Synthesis of Pyrazolo[1,5-a]pyrimidines ofGeneral Formula (I)]

a) An intimate mixture of the Boc protected intermediate (II) (100 mg)and trans-1, 4-cyclohexanediamine (1.5 g) were heated together at 80-85°C. for 90 min, then cooled. The crude material was then partitionedbetween dichloromethane and saturated NaHCO₃ solution. The organic phaseis then separated and washed with water. Dried over MgSO⁴ andconcentrated in vacuo. The crude material dissolved in dichloromethane(10 ml) and trifluoroacetic acid (5 ml). Stirred for 1 h at roomtemperature, then evaporated in vacuo. The residue was partitionedbetween saturated NaHCO₃ and dichloromethane, the organic phase wasseparated, dried over MgSO₄ then subjected to column chromatography oversilica gel. Eluent dichloromethane, then gradient elution up to 95%dichloromethane+5% (10 M NH₃ in methanol). Typical purified yield 20 mg

b) An intimate mixture of the Boc protected intermediate (II) (100 mg)and trans-1, 4-cyclohexanediamine (1.5 g) were heated together at 80-85°C. for 18 hr then cooled. The crude material was then partitionedbetween dichloromethane and saturated NaHCO₃ solution. The organic phaseis then separated and washed with water. Dried over MgSO₄ andconcentrated in vacuo. The crude product subjected to columnchromatography over silica gel. Eluent dichloromethane, then gradientelution up to 95% dichloromethane+5% (10 M NH₃ in methanol). Typicalpurified yield 20 mg.

c) The intermediate (II) (0.1 mmol) was dissolved in toluene (1 ml) andthe amine (1.2 equivalents) was added.Tris(dibenzylideneacetone)dipalladium (0) (2 mol %),2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (4 mol %) and sodiumtert-butoxide (1.2 equivalents) were added sequentially under anatmosphere of nitrogen. The reaction was heated and agitated overnightat 80° C. following which the reaction was filtered through a 0.45micron filter. The solvent was removed in vacuo and the residue wasresuspended in dichloromethane (2 ml). Trifluoroacetic acid (0.8 ml) wasadded and the reactions allowed to stand for 1 h at room temperature.The mixture was evaporated to dryness, in vacuo, and the resultantresidue was dissolved in N,N-dimethylformamide (1 ml), filtered andpurified by prep-HPLC to give the product (I). (Analysisperformed—LC/MS.)

d) Further elaborations of compounds of General Formula (I)

i) Acylations with acid halides, sulfonyl halides, isocyanates andisothicyanates

To a solution of Compound 2 (50 mgs) in dichloromethane (10 ml) wasadded triethylamine (1.1 equivalents) followed by the dropwise additionof the acid halide, sulfonyl halide, isocyanate or isothicyanate (1.05equivalents). The mixture was stirred for 1-2 hours the washed withwater, dried over MgSO₄, the solvent was removed in vacuo then theresidue subjected to column chromatography over silica gel. Eluentdichloromethane, then gradient elution up to 95% dichloromethane+5% (10M NH₃ in methanol) to afford compound, for example Mp (° C.)/or forCompound No NR⁵R⁶ gums M⁺, M⁻ 25 trans-4-Acetylamino-c- 239-241(d)hexylamine 27 trans-4-Methylsulphonylamino-c- Gum, 453, 451 hexylamine38 trans-4-MeNHCONH-c- 233-238 hexylamine 57 trans-4-MeNHCSNH-c- 167-169hexylamineii) Reductive Aminations

To a solution of Compound 2 (50 mgs) in tetrahydrofuran (5 ml) was addedcyclohexanone (1.1 equivalents) and the reaction was heated overnight at60° C. To the cooled mixture was then added sodium cyanoborohydride (5equivalents) and stirred at ambient temperature for 2 hours. The mixturewas evaporated to dryness, in vacuo, and the resultant residue dissolvedin water and ethyl acetate. The organic phase was separated, dried overMgSO₄ then subjected to column chromatography over silica gel. Eluentdichloromethane, then gradient elution up to 95% dichloromethane+5% (10M NH₃ in methanol) to afford compound 134, mp 85-87° C., 20 mg

Compounds of general formula (I) prepared by the above procedures arerecorded in Table B. The numbers assigned to each of the compounds inTable B correspond to the Compound Nos. of the compounds listed asspecific examples in Table A above. Compounds were characterised by massspectrometry using single quadrupole instrumentation with anelectrospray source. M+H indicates values obtained for compoundmolecular mass (M) with proton (H) capture and M-H compound molecularmass (M) with proton (H) loss. Melting points (mp) are uncorrected; (d)denotes decomposition at or near the melting point. Compounds which werenot solids were gums. TABLE B ESI/MS Compound No. Mp (° C.) M + H M − H1 171-173 389 2 144-146(d) 375 373 3 gum 391 4 140-143(d) 403 5138-141(d) 387 6 gum 371 7 gum 357 8 152-155(d) 9 175-177 337 10 88-89(d) 323 11  89-92(d) 357 12 158-161(d) 357 355 13 153-156(d) 35714 gum 371 15 gum 366 16 gum 341 339 17 gum 353 351 18 gum 375 373 19gum 363 364 20 141-144(d) 391 389 21 gum 415 413 22 gum 359 357 23 96-98(d) 24  97-102(d) 25 239-241(d) 417 415 26 201-201(d) 368 27 gum453 451 28 211-214(d) 479 477 29 131-134 351 30 Gum 379 377 31 135-138367 32 Gum 348 346 33 Gum 365 34 Gum 379 35 183-186 367 36 181-183 365363 37  87-92(d) 365 363 38 233-238 431 430 39 Gum 407 40 114-118(d) 449447 41  85-90(d) 449 42 Gum 483 481 43  87-92(d) 351 44  68-72(d) 337 45251-254(d) 351 46  77-81(d) 415 47  78-82(d) 353 48 Gum 287 49 227-228494 50 Gum 376 51 Gum 367 365 52 158-162(d) 429 427 53 192-194 461 46354  92-96(d) 367 365 55  94-98(d) 403 56  89-91 375 57 167-169 448 44658 Gum 301 299 59 Gum 285 60 Gum 391 389 61 gum 375 373 62  64-66 353351 63  62-65 407 405 64  80-83 429 427 65  86-88 66 129-130 67 163-16768  95-100 69 217-219 70 184-188 339 337 71 gum 371 72 175-177 73 gum347 74 gum 361 75 175-177 76  95-100 77  85-90 78 gum 349 79  80(d) 80149-150 335 81 230-232 347 82 218-219 347 83  90-100 84 164-166 85166-168 86 gum 335 87 gum 389 88 105-106 361 359 89 172-173 403 401 90Gum 338 91 100-105 425 423 92 130-140 348 346 93 100-105 391 389 94 Gum335 333 95 155-157 361 359 96  55-57 339 337 97  60-63 339 337 98  60-62415 413 99 Gum 387 385 100  66-71 363 361 101  88-91 449 447 102 120-123401/403 399/401 103 216-219 413 411 104 155-157 395 393 105 Gum 353 351106  95-97 435 433 107 Gum 441 108 106-110 375 109  98-106 359 357 110103-106 361 359 111 Gum 313 112 119-121 371 369 113 150-153 399 397 114178-180 367 115  80-82 383 381 116 Gum 321 319 117  69-71 387 385 118120-130 387 385 119  52-54 491 489 120 Gum 465 463 121 Gum 479 122 Gum521 519 123 116-120 532 124  58-61 493 491 125 207-210 445 443 126 65-69 471 469 127 Gum 459 457 128  48-51 455 453 129  60-70 471 469 130Gum 459 457 131 Gum 505 132 Gum 519 133  72-74 596 134  85-87 455 135133-135 415 136 Gum 470 137  55-60 458 138 Gum 531 139 121-124 353 351140 130-134 435 433 141 202-204 327 325MAPKAP-Kinase 2 Assay[Compound Preparation]

Compounds are dissolved in DMSO at a concentration of 3 mM and stored inaliquots at −20° C. Compounds in DMSO from these stock aliquots arediluted in 30% DMSO to produce initial working stock solutions of 1 mMand 3 mM. Both of these stock solutions are then subjected to 1:10serial dilutions in 30% DMSO in order to prepare 3000, 1000, 300, 100,30, 10, 3, 1, 0.1, 0.01 μM stock solutions. 5 μl of each stock solutionis used per 50 μl reaction to give final assay concentrations of 300,100, 30, 10, 3, 1, 0.3, 0.1, 0.01, 0.001 μM.

[Assay]

The kinase reaction is conducted in a round-bottomed polypropylene96-well plate. MAPKAP-K2 is diluted to 25 mU/μl in diluent buffer (50 mMTris/HCl. pH7.5, 0.1 mM EGTA, 0.1% (v/v) β-mercaptoethanol, 1 mg/mlBSA). 5 μl compound or 30% DMSO is added to each well followed by 25 μlsubstrate cocktail (10 μM ATP, 30 μM peptide (KKLNRTLSVA), 0.5 μCi³³P-γ-ATP in 50 mM Tris pH7.5, 0.1 mM EGTA, 10 mM Mg-acetate, 0.1% BME).The reaction is initiated with the addition of 20 μl enzyme solution perwell or 20 μl diluent buffer without enzyme. The plate is shaken for 10sec and then left at room temperature for 30 min. The reaction isterminated with 50 μl 150 mM phosphoric acid. 90 μl of the reactionmixture is then transferred into a 96-well P81 filter plate (Millipore)and incubated at room temperature for 5 min. The filter plate is thenwashed 4 times with 200 μl 75 mM phosphoric acid per well on a platevacuum manifold (Millipore) and dried in an oven for 2-3 h. PackardMicroScint ‘0’ (30 μl) is then added to each well, the plate is mixedfor 30 min and subjected to liquid scintillation counting on a PackardTopCount.

[Interpretation]%Control=(X−B)/(Tot−B)×100%Inhibition=100−% ControlX=cpm of the test compound wellsB=cpm of wells without enzymeTot=cpm of wells with DMSO vehicle only

The efficacy of the compounds in Table B against kinases is shown inTable C. (The activity is presented as +, ++, or +++ representingactive, more active and very active based on assays conducted attypically 1-100 μM.) TABLE C MAPKAP-K2 Compound No activity 1 ++ 2 +++ 3+++ 4 ++ 5 +++ 6 +++ 7 ++ 8 ++ 9 +++ 10 +++ 11 +++ 12 +++ 13 +++ 14 ++15 +++ 16 +++ 17 +++ 18 + 19 +++ 20 +++ 21 +++ 22 +++ 23 + 24 + 25 + 26+++ 27 + 28 + 29 ++ 30 ++ 31 + 32 +++ 33 ++ 34 ++ 35 +++ 36 +++ 37 +++38 + 39 ++ 40 +++ 41 +++ 42 +++ 43 +++ 44 +++ 45 ++ 46 +++ 47 ++ 48 ++49 + 50 + 51 +++ 52 +++ 53 ++ 54 ++ 55 ++ 56 ++ 57 + 58 +++ 59 +++ 60+++ 61 + 62 +++ 63 +++ 64 +++ 65 +++ 66 +++ 67 +++ 68 +++ 69 +++ 70 +++71 ++ 72 ++ 73 ++ 74 +++ 75 +++ 76 +++ 77 +++ 78 + 79 ++ 80 + 81 + 82 +83 +++ 84 +++ 85 +++ 86 ++ 87 +++ 88 ++ 89 +++ 90 ++ 91 +++ 92 +++ 93 ++94 + 95 + 96 ++ 97 +++ 98 +++ 99 +++ 100 ++ 101 +++ 102 +++ 103 ++ 104+++ 105 +++ 106 +++ 107 +++ 108 +++ 109 +++ 110 ++ 111 ++ 112 +++ 113+++ 114 ++ 115 +++ 116 + 117 + 118 ++ 119 ++ 120 + 121 ++ 122 ++ 123 ++124 ++ 125 ++ 126 + 127 ++ 128 ++ 129 ++ 130 + 131 ++ 132 ++ 133 + 134++ 135 ++ 136 +++ 137 + 138 + 139 +++ 140 +++ 141 +++

INDUSTRIAL APPLICABILITY

The Pyrazolo[1,5-a]pyrimidine derivatives represented by formula I andtheir pharmaceutically acceptable salts exhibit excellent kinaseinhibiting activity particularly MAPKAP-K2 inhibiting activity). Drugscomprising the compounds as effective ingredients are therefore expectedto be useful as therapeutic or prophylactic agents for a protein kinasemediated disorder in which kinase is implicated, such as such asinflammatory disease, autoimmune disease, destructive bone disorder,cancer and/or tumour growth.

1. A use of a compound of formula (I):

wherein R¹ is hydrogen R² is hydrogen R³ is C1-C8 optionally substitutedalkyl, C2-C8 optionally substituted alkenyl, C2-C8 optionallysubstituted alkynyl, C3-C8 optionally substituted cycloalkyl, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted arylalkyl, optionally substituted heteroarylalkyl,optionally substituted arylalkenyl, optionally substitutedheteroarylalkenyl, optionally substituted arylalkynyl, or optionallysubstituted heteroarylalkynyl; R⁴ is hydrogen; R⁵ is C1-C8 optionallysubstituted alkyl, C2-C8 optionally substituted alkenyl, C2-C8optionally substituted alkynyl, C3-C8 optionally substituted cycloalkyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted arylalkyl, optionally substitutedheteroarylalkyl, optionally substituted arylalkenyl, optionallysubstituted heteroarylalkenyl, optionally substituted arylalkynyl, oroptionally substituted heteroarylalkynyl, optionally substitutedheterocyclyl or optionally substituted heterocyclylalkyl; R is hydrogen,C1-C8 optionally substituted alkyl, C2-C8 optionally substitutedalkenyl, C2-C8 optionally substituted alkynyl or C3-C8 optionallysubstituted cycloalkyl; or R⁵ and R⁶ together may be taken together withthe nitrogen to which they are attached to form a mono or bicyclicheterocycle with 5-7 members in each ring and optionally containing, inaddition to the nitrogen, one or two additional heteroatoms selectedfrom N, O and S, the said mono or bicyclic heterocycle may optionally besubstituted with one or more substituents; or pharmaceuticallyacceptable salts, or other pharmaceutically acceptable biohydrolyzablederivatives thereof, including esters, amides, carbamates, carbonates,ureides, solvates, hydrates, affinity reagents or prodrugs thereof, inthe manufacture of a medicament for use in inhibiting protein kinases.2. The use as claimed in claim 1, wherein R³ is C1-C8 optionallysubstituted alkyl, C2-C8 optionally substituted alkenyl, C2-C8optionally substituted alkynyl, C3-C8 optionally substituted cycloalkyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted arylalkyl or optionally substitutedheteroarylalkyl.
 3. The use as claimed in claim 2, wherein R³ is C2-C8optionally substituted alkenyl, optionally substituted aryl oroptionally substituted arylalkyl.
 4. The use as claimed in any one ofclaims 1 to 3, wherein R⁵ is C1-C8 optionally substituted alkyl, C2-C8optionally substituted alkenyl, C2-C8 optionally substituted alkynyl,C3-C8 optionally substituted cycloalkyl, optionally substitutedheterocyclyl or optionally substituted heterocyclylalkyl.
 5. The use asclaimed in claim 4, wherein R⁵ is C3-C8 cycloalkyl substituted by NHR⁷,wherein R⁷ is optionally substituted heterocyclyl or optionallysubstituted heterocyclylalkyl.
 6. The use as claimed in any one ofclaims 1 to 5, wherein R⁶ is hydrogen or C1-C8 optionally substitutedalkyl.
 7. The use as claimed in claim 6, wherein R⁶ is hydrogen.
 8. Theuse as claimed in any one of claims 1 to 7, wherein the medicament isfor use as an inhibitor of MAPKAP-K2.
 9. The use as claimed in claim 8,wherein the medicament is for use in the prevention or treatment of aMAPKAP-K2-mediated disorder.
 10. The use as claimed in claim 9, whereinthe MAPKAP-K2 mediated disorder is a neurological disorder (includingdementia), an inflammatory disease, a disorder linked to apoptosis,particularly neuronal apoptosis, stroke, sepsis, autoimmune disease,destructive bone disorder, proliferative disorder, cancer, infectiousdisease, allergy, ischemia reperfusion injury, heart attack, angiogenicdisorder, organ hypoxia, vascular hyperplasia, cardiac hypertrophy,thrombin induced platelet aggregation.
 11. The use as claimed in claim10, wherein the disorder is a neurodegenerative disorder.
 12. The use asclaimed in claim 11, wherein the neurodegenerative disorder is dementia,Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis,Huntington's disease, senile chorea, Sydenham's chorea, hypoglycemia,head and spinal cord trauma including traumatic head injury, acute andchronic pain, epilepsy and seizures, olivopontocerebellar dementia,neuronal cell death, hypoxia-related neurodegeneration, acute hypoxia,glutamate toxicity including glutamate neurotoxicity, cerebral ischemia,dementia linked to meningitis and/or neurosis, cerebrovascular dementia,or dementia in an HIV-infected patient.
 13. The use as claimed in claim10, wherein the disorder results from inflammation.
 14. The use asclaimed in claim 13, wherein the disorder is inflammatory boweldisorder, bronchitis, asthma, acute pancreatitis, chronic pancreatitis,allergies of various types or Alzheimer's disease.
 15. The use asclaimed in claim 10, wherein the disorder is an autoimmune disease. 16.The use as claimed in claim 15, wherein the autoimmune disease isrheumatoid arthritis, systemic lupus erythematosus, glomerulonephritis,scleroderma, chronic thyroiditis, Graves's disease, autoimmunegastritis, diabetes, autoimmune haemolytis anaemia, autoimmuneneutropaenia, thrombocytopenia, atopic dermatitis, chronic activehepatitis, myasthenia gravis, multiple sclerosis, ulcerative colitis,Crohn's disease, psoriasis or graft vs host disease.
 17. A method oftreating or preventing a MAPKAP-K2-mediated disorder, which comprisesadministering to said individual at least one compound as defined in anyone of claims 1 to 7 or the composition defined in claim 8 or claim 9.18. The method as claimed in claim 17, wherein the MAPKAP-K2 mediateddisorder is a neurological disorder (including dementia), aninflammatory disease, a disorder linked to apoptosis, particularlyneuronal apoptosis, stroke, sepsis, autoimmune disease, destructive bonedisorder, proliferative disorder, cancer, infectious disease, allergy,ischemia reperfusion injury, heart attack, angiogenic disorder, organhypoxia, vascular hyperplasia, cardiac hypertrophy, thrombin inducedplatelet aggregation.
 19. The method as claimed in claim 18, wherein thedisorder is a neurodegenerative disorder.
 20. The method as claimed inclaim 19, wherein the neurodegenerative disorder is dementia,Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis,Huntington's disease, senile chorea, Sydenham's chorea, hypoglycemia,head and spinal cord trauma including traumatic head injury, acute andchronic pain, epilepsy and seizures, olivopontocerebellar dementia,neuronal cell death, hypoxia-related neurodegeneration, acute hypoxia,glutamate toxicity including glutamate neurotoxicity, cerebral ischemia,dementia linked to meningitis and/or neurosis, cerebrovascular dementia,or dementia in an HIV-infected patient.
 21. The method as claimed inclaim 18, wherein the disorder results from inflammation.
 22. The methodas claimed in claim 21, wherein the disorder is inflammatory boweldisorder, bronchitis, asthma, acute pancreatitis, chronic pancreatitis,allergies of various types or Alzheimer's disease.
 23. The method asclaimed in claim 18, wherein the disorder is an autoimmune disease. 24.The method as claimed in claim 23, wherein the autoimmune disease isrheumatoid arthritis, systemic lupus erythematosus, glomerulonephritis,scleroderma, chronic thyroiditis, Graves's disease, autoimmunegastritis, diabetes, autoimmune haemolytis anaemia, autoimmuneneutropaenia, thrombocytopenia, atopic dermatitis, chronic activehepatitis, myasthenia gravis, multiple sclerosis, ulcerative colitis,Crohn's disease, psoriasis or graft vs host disease.
 25. The method asclaimed in any one of claims 18 to 24 wherein one or more active agentsis/are administered to the individual simultaneously, subsequently orsequentially to administering the compound.
 26. A method for determiningthe activity of the compounds as defined in any one of claims 1 to 7,comprising providing a system for assaying the activity and assaying theactivity of a compound as defined in any of claims 1 to
 7. 27. Themethod as claimed in claim 26 wherein the assay is for the proteinkinase inhibiting activity of the compound.
 28. A method of inhibitingthe activity or function of a protein kinase, which comprises exposing aprotein kinase to a compound as defined in any of claims 1 to
 7. 29. Amethod of inhibiting the activity or function of MAPKAP-K2, whichcomprises exposing MAPKAP-K2 to a compound as defined in any of claims 1to
 7. 30. The method as claimed in claim 29, which is performed in aresearch model, in vitro, in silico, or in vivo.